RNA Isolation Procedures for Rotavirus see Note

2.1.1. Inactivation of Endogenous Ribonucleases (see Note 2)

1. Diethyl-pyrocarbonate (DEPC).

2. RNase AWAY (Molecular Bioproducts, San Diego, CA).

2.1.2. RNA Extraction: SDS/Proteinase K Digestion-Phenol Chloroform Extraction

1. 10 mg/mL Proteinase K, prepared using DEPC water.

2. 10% (w/v) Sodium-dodecyl sulfate (SDS); prepared using DEPC water.

3. Chloroform.

4. Absolute ethanol.

5. 1:1 Phenol/chloroform (see Note 3).

6. Sterile 1.5-mL Eppendorf tubes.

Fig. 3. Schematic representation of the seminested PCR assay that identifies the G-type of a rotavirus isolate. The locations of the variable regions on gene segment 9 are indicated. PCR primers identify distinct serotypes b y amplifying PCR products of defined length (see also Table 1 for primer sequences).

Fig. 3. Schematic representation of the seminested PCR assay that identifies the G-type of a rotavirus isolate. The locations of the variable regions on gene segment 9 are indicated. PCR primers identify distinct serotypes b y amplifying PCR products of defined length (see also Table 1 for primer sequences).

2.1.3. Removal of Contaminating Genomic DNA (see Note 4)

2. 3:1 Phenol/chloroform.

3. 3 M Sodium acetate.

4. DNase 1 (RNase free; Roche Diagnostics, East Sussex, UK).

5. 10X Tris buffer: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl2.

6. Nuclease free water (Promega, Madison, WI).

2.2. Electropherotying of Rotavirus RNA Genomes

2.2.1. Polyacrylamide Gel Electrophoresis (PAGE)

1. 38% (w/v) Acrylamide/bisacrylamide mix: prepared by dissolving 74 g of acrylamide (molecular biology grade) and 2 g bis-acrylamide (molecular biology grade) in 200 mL DEPC-water. Store in a dark bottle at room temperature.

4. 10% (w/v) Ammonium persulfate (Sigma, Poole, UK).

6. TEMED (N,N,N',W-Tetramethylethylenediamine; Sigma).

7. Isobutanol.

8. 5X Tris-glycine buffer: 25 mM Tris-HCl, 250 mM glycine, pH 8.3, 0.1% (w/v) SDS.

9. Loading buffer: 62 mM Tris, 2% (w/v) SDS, 0.001% (w/v) bromophenol blue, 10% (v/v) glycerol.

2.2.2. Staining and Detection of Polyacrylamide Gels

1. Buffer 1: 10% (v/v) ethanol, 0.5% (v/v) glacial acetic acid.

2. 0.011 M Silver stain.

3. Buffer 2: 0.75 M NaOH containing 0.1 M formaldehyde and 0.0023 M sodium boro-hydride.

4. Buffer 3: 0.07 M sodium carbonate.

2.3. PCR Amplification Methods

1. 100 mM Stocks of each deoxyribonucleoside triphosphate (dNTP) dATP, dCTP, dGTP and dTTP (Promega).

2. dNTP working stock solutions containing 1.25 mM of each dNTP, prepared by diluting 2.5 ^L of each dNTP in 190 ^L of sterile water.

3. 10X PCR buffer: 100 mM Tris-HCl, pH 9.0, 500 mM KCl (Promega).

5. 5 U/mL Taq DNA Polymerase (Promega).

6. AMV (avian myeloblastosis virus)-reverse transcriptase (AMV-RT; Promega).

7. 5X AMV buffer: 250 mM Tris-HCl, pH 8.3, 250 mM KCl, 50 mM MgCl2, 2.5 mM spermidine, 50 mM dithiotreitol (DTT).

9. Dimethyl sulphoxide (DMSO).

10. RnasinĀ® ribonuclease inhibitor (Promega).

11. Sterile mineral oil (Sigma).

12. Sterile water.

13. Oligonucleotide primers (Table 1) were adjusted to the required concentrations. (All primers were obtained from Oswel Scientific and purified by high-performance liquid chromatography prior to use.)

2.4. Purification of PCR Products

1. PCR purification kit (QIAGEN, West Sussex, UK).

2.5. Conventional Agarose Gel Electrophoresis

1. Agarose (Promega).

2. 10X Tris-acetate EDTA (TAE): 40 mM Tris-HCl, pH 7.8, 40 mM glacial acetic acid, 2 mM EDTA.

3. 10X Gel loading buffer: 6.6 mL glycerol, 3.3 mL 10X TAE, 100 ^L of 10% (w/v) bro-mophenol blue.

4. Ethidium bromide (10 mg/mL; Sigma).

5. DNA molecular weight markers: a range of DNA markers are commercially available (Roche Diagnostics) that have various fragment molecular weight ranges. In this study three DNA markers were used, including DNA marker XIV, which has 16 fragments that range in size from 100 to 1500 bp with an additional band at 2642 bp; DNA marker III providing 13 fragments with sizes ranging from 0.12 to 21.2 kbp; and DNA marker V, which contains 22 fragments ranging in sizes from 8 to 587 bp.

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